Herpesviridae is a large family of enveloped linear dsDNA-containing animal viruses. Herpesviruses are morphologically similar. The virion (˜120-200 nm diam.) contains a core (DNA wound around a central protein structure) within an icosahedral capsid (˜100-110 nm diam.) comprising 12 pentameric and 150 hexameric capsomers. The viron is enclosed by a lipoprotein envelope bearing surface projections. The linear dsDNA genome characteristically contains repeated terminal and/or internal sequences.
Herpesviruses have been isolated from a wide range of animals, including mammals, birds, reptiles, amphibians, and fish. Many herpesviruses cause disease in their primary host(s), and may remain latent within the tissue of the host, often for life. Virus transmission commonly occurs by direct contact of mucosal surfaces. Some herpesviruses can be transmitted via body fluids (e.g., milk; via the placenta; etc).
Herpes simplex virus (HSV) type 1 or 2 is a causative agent for serious infections in humans. Herpes simplex diseases are characterized by the formation of thin-walled vesicles, which ulcerate, crust and heal; the vesicles occur, often in clusters, on skin and/or mucous membranes. Transmission occurs as a result of close physical contact; e.g., sexual contact, kissing, close contact sports such as wrestling (Herpes gladiatorum). HSV incubation periods range from 2-12 (average 6) days. The disease state varies from subclinical to severe, and is occasionally fatal. HSV can remain latent in nerve cells near the site of infection. Reactivation may occur spontaneously or in response to other infections (e.g., stress, immunosuppression). In neonates and immunodeficient individuals, HSV may become-disseminated; often affecting the liver, adrenal glands, brain, etc.
HSV-2 is associated with genital, and hence neonatal, infections (Herpes genitalis), and is a disease of significant morbidity in infected individuals (Whitley, 1996). In addition to the conditions described above, other symptoms may include e.g. fever, dysuria, pain, and malaise. In women, the cervix is often the main site of genital infection (herpetic cervicitis). HSV-2 infection in women is associated with an increased risk of abortion and of cervical cancer. Individuals with active HSV-2 have an increased risk of acquiring HIV if exposed to the virus (Augenbraun and McCormack).
Neonatal herpes is usually acquired (during birth) from a mother infected with HSV-2. Fatality rates may be 50% or more in untreated cases. Surviving infants commonly show neurological and/or ocular secondary disorders.
Clinical diagnosis of HSV-2 infection is established by microscopic examination of lesion samples, or biopsies from e.g. skin, brain or liver for multinucleate giant cells with eosinophilic intranuclear inclusion bodies, or by various immunofluorescence techniques (e.g., ELISA).
A number of antiviral agents (e.g. vidarabine, acyclovir, IDU and trifluorothymidine) have activity against HSV and may be effective in some cases (e.g. vidarabine is used against HSV encephalitis). These drugs are not generally effective in preventing recurrence or transmission, however. There remains an unmet need for an effective vaccine against HSV-2 to induce protective immunity and to prevent or to reduce primary infection and, ideally, to reduce recurrent disease and transmission.
Numerous approaches have been attempted to obtain immunization against HSV infection (e.g., glycoprotein subunits, inactivated virus, attenuated virus, and various HSV antigens. See Krause and Straus, 1999; Bernstein and Stanberry, 1999; and Stanberry, 1998). These approaches have shown little to no effectiveness, however (e.g., HSV glycoprotein subunit vaccines, Corey et al., 1999 and Straus et al, 1997; attenuated HSV, Cadoz, et al., 1992).
The use of replication-defective mutant viral strains is a promising avenue of induced immunization against HSV in animal models (Boursnell et al., 1997; Da Costa et al., 1997; Farrell et al., 1994; McLean et al., 1994; McLean, 1996; Morrison and Knipe, 1994; Morrison, Da Costa, and Knipe, 1998; Nguyen et al., 1992; and Stanberry, 1999). Current studies, however, use single mutant viruses, which carry with it the threat of back mutation (reversion to a virulent wild type). Standard vaccine design typically utilizes strains with two or more (non-reverting) mutations to increase safety of the vaccine (Curtiss et al., 1994).
In an effort to develop a live mutant virus vaccine, while reducing the risk of reversion, Da Costa et al. have developed a double deletion mutant HSV-2 strain. This strain lacks two genes essential for DNA replication, thus rendering the mutant incapable of DNA synthesis and viral replication. This double deletion mutant virus strain fails to form plaques or to give any detectable single cycle yields in normal monkey or human cells, yet it is capable of eliciting an immune response (i.e., it functions as an effective immunogen). This double deletion mutant HSV-2 strain induces antibody titers in mice equivalent to those induced by single deletion mutant viruses (Da Costa et al., manuscript submitted).
Because this double deletion mutant HSV-2 strain is replication defective, the replication gene product components it lacks must be provided. There is a need, therefore, for a cell line capable of complementing this double deletion mutant HSV-2 strain, enabling the propagation of the strain, thus providing vaccine production level growth stock of the mutant strain.